Distinct forebrain regions define a dichotomous astrocytic profile in multiple system atrophy

The growing recognition of a dichotomous role of astrocytes in neurodegenerative processes has heightened the need for unraveling distinct astrocytic subtypes in neurological disorders. In multiple system atrophy (MSA), a rare, rapidly progressing atypical Parkinsonian disease characterized by increased astrocyte reactivity. However the specific contribution of astrocyte subtypes to neuropathology remains elusive. Hence, we first set out to profile glial fibrillary acidic protein levels in astrocytes across the human post mortem motor cortex, putamen, and substantia nigra of MSA patients and observed an overall profound astrocytic response. Matching the post mortem human findings, a similar astrocytic phenotype was present in a transgenic MSA mouse model. Notably, MSA mice exhibited a decreased expression of the glutamate transporter 1 and glutamate aspartate transporter in the basal ganglia, but not the motor cortex. We developed an optimized astrocyte isolation protocol based on magnetic-activated cell sorting via ATPase Na+/K+ transporting subunit beta 2 and profiled the transcriptomic landscape of striatal and cortical astrocytes in transgenic MSA mice. The gene expression profile of astrocytes in the motor cortex displayed an anti-inflammatory signature with increased oligodendroglial and pro-myelinogenic expression pattern. In contrast, striatal astrocytes were defined by elevated pro-inflammatory transcripts accompanied by dysregulated genes involved in homeostatic functions for lipid and calcium metabolism. These findings provide new insights into a region-dependent, dichotomous astrocytic response—potentially beneficial in the cortex and harmful in the striatum—in MSA suggesting a differential role of astrocytes in MSA-related neurodegenerative processes. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-023-01699-3.


Introduction
Astrocytes represent a highly heterogeneous glial population in the central nervous system (CNS) with diverse functions related to maintaining the integrity of the blood-brain barrier and the cerebral homeostasis of fluids, ions, pH, and neurotransmitters [1,2].Astrocytes contribute to protecting from glutamate-mediated excitotoxicity by removing glutamate from the synaptic cleft and preventing hyperstimulation of postsynaptic receptors [3,4].They also closely interact with neurons as well as blood vessels thereby regulating CNS metabolism [5] and, by storing and release of glycogen, transfer of glucose metabolites and generation of lactate, dynamically support neural functions [6][7][8].Furthermore, astrocytes regulate lipid metabolism such as cholesterol efflux and oxidation of fatty acids [9,10].Besides regulating essential processes in the CNS, astrocytes have been described to closely interact with microglia and respond dynamically to cytokines secreted [11,12].This astro-microglial crosstalk was shown to result in conversion of astrocytes into a neurotoxic or neuroprotective state dependent on the stimuli (Fig. 2A) [11,13].
For decades, expression of glial fibrillary acidic protein (GFAP), an astrocytic-specific marker in the brain, is used to define astrocytic identity and reactivity [1].Expression of GFAP, however, varies among astrocyte subpopulations and is region-dependent; for example, grey matter regions demonstrate lower GFAP levels compared to white matter and hippocampus [14].Therefore, using ubiquitously expressed astrocyte markers such as aldehyde dehydrogenase 1 family member L1 (ALDH1L1), aldolase C, or glutamine synthetase (GS) are important for a comprehensive characterization of astrocytic populations [1].
Rapid methodological developments resulted in the identification of transcriptional astrocytic expression profiles in physiological as well as pathological states [15][16][17].These assignments led to the translation from structural phenotyping to region-and context-specific functions thereby identifying astrocytic subpopulations with a spectrum of potential effector functions ranging from neurotoxic properties with impaired autophagy, synapse elimination, and dopamine regulation [18,19] to neuroprotective signatures promoting CNS recovery and repair by secreting transforming growth factor β (TGF-β) and brain derived neurotrophic factor (BDNF), angiopoietin-1 (ANG1), and expression of connexin-43 (CX43) [20][21][22].
Multiple system atrophy (MSA), a rare sporadic oligodendroglial α-synucleinopathy, is a rapidly progressing neurodegenerative disease with an average survival rate of 6-9 years [23][24][25].Clinically, MSA is subdivided into a parkinsonian (MSA-P) and cerebellar phenotype (MSA-C) accompanied by an early and severe autonomic failure [26].In MSA-P, the dorsolateral putamen, caudate nucleus, and substantia nigra display pronounced neuronal loss whereas cortical areas such as the precentral gyrus are less affected [27].Reactive astrogliosis has been shown in the putamen of MSA patients demonstrating an inverse correlation between reactive astrocytes and the distance to glial cytoplasmic inclusions [28,29].
To shed more light into astrocytic reactivity in MSA, we first examined post mortem prototypical MSA-P brain regions to determine reactive astrogliosis in the cortex, the striatum, and the substantia nigra.Next, to test if astrocyte reactivity observed in post mortem tissue is recapitulated in a mouse model of MSA, we took advantage of the MBP29-hα-syn mouse model.
In 2005, Shults and colleagues generated a mouse model overexpressing human α-synuclein (hα-syn) in oligodendrocytes under the control of a myelin-basicprotein (MBP) specific promotor (MBP29-hα-syn) [30].These mice start to develop progressive motor deficits at the age of 10 weeks, prematurely die between 20 and 30 weeks of age, and show a severe neuronal loss accompanied by a widespread demyelination and region-specific microgliosis matching important functional and neuropathological features of MSA [31,32].Due to the important role of astrocytes to alter neurodegenerative and inflammatory processes [33][34][35] there is an urgent need to comprehensively characterize astrocytic subpopulations in MSA and its respective model for identifying potential targets suitable for future interventional strategies.After confirming a similar astrocytic phenotype in MBP29-hα-syn mice, we made use of an optimized stateof-the-art isolation protocol using magnetic activated cell sorting (MACS) approach to decipher the transcriptional profile of astrocytes in CNS regions differentially affected by the oligodendroglial synucleinopathy.

Human post mortem brain tissue
Post mortem cortex, putamen and substantia nigra of MSA-P patients and age-and sex-matched controls (each n = 4, Table 1) were obtained from the Netherlands Brain Bank (NBB), Netherlands Institute for Neuroscience, Amsterdam (open access: http:// www.brain bank.nl).MSA cases were clinically diagnosed and neuropathologically confirmed as MSA-P according to the second consensus criteria (Table 1; [36]).Paraffin-embedded tissue of the cortex, the striatum, and the substantia nigra were sectioned at a thickness of 5 µm.

Animals
Transgenic MBP29-hα-syn mice overexpressing human α-syn under the control of the murine MBP promoter and corresponding non-transgenic littermates (NTG) serving as controls were anesthetized and sacrificed at an age of 4 weeks.For immunofluorescence staining, animals were perfused using 4% paraformaldehyde (PFA) and 0.9% sodium chloride solution according to the European and National Institute of Health guidelines for humane treatment.Afterwards, brains were transferred into 4% PFA for 2-4 h and subsequently stored in 30% sucrose solution.Whole forebrains were sagittally sectioned at a thickness of 40 µm and stored in cryoprotection solution (25% 0.2 mol/L phosphate buffer) at − 20 °C until further staining.For expression analysis of proteins and mRNA, by Western blot (WB) and quantitative PCR (qPCR), respectively, mice were perfused using phosphate-buffered saline (PBS).After the removal of the brain, the cortices and striata were subsequently micro-dissected, snap-frozen in liquid nitrogen, and stored at −80 °C until homogenization.For the isolation of astrocytes and oligodendrocytes, mice were sacrificed, brains were removed, dissected as described above and stored at 4 °C in Dulbecco's Phosphate Buffered Saline containing MgCl 2 and CaCl 2 (D-PBS) until further processing for MACS.

Immunohistochemistry of human post mortem tissue
Immunohistochemistry on formalin-fixed and paraffinembedded tissue was performed following a previously published protocol [31].In brief, sections at a thickness of 5 µm were deparaffinized.After microwave antigen retrieval using citrate buffer, sections were stained using the REAL EnVision Detection System peroxidase/DAB+, Rabbit/Mouse (Dako, # K5007) according to the manufacturer's protocol.The kit uses 3,3′-diaminobenzidine tetrahydrochloride (DAB) as a chromogen.Finally, sections were counterstained using hematoxylin.Primary antibodies and staining conditions were as follows: antigen retrieval using citrate buffer (0.1 M) for 30 min followed by anti-GFAP (Agilent Cat# M0761, RRID:AB_2109952, 1:100) incubation overnight.As positive control, an intracerebral metastasis of a breast carcinoma with an adjacent reactive border zone was used.Analyzed areas of each region is given in Additional file 1: Table S2.

Isolation of murine astrocytes and oligodendrocytes
Adult astrocytes and oligodendrocytes were isolated using MACS.
For proper labeling, cells were incubated with antibodies at 4 °C for 15 min.Pellets were resuspended in cold FC buffer and dead cells were stained using DAPI.ACSA2 + and O4 + cells were subsequently analyzed using the BD LSRFortessa ™ Cell Analyzer (BD Biosciences GmbH).Quantification of ACSA2 + and O4 + populations were calculated using FlowJo ™ Software (BD Biosciences GmbH).

RNA isolation
RNA was isolated using the RNeasy Plus Micro Kit (74034, Qiagen).Cells were lysed in RLT Plus Buffer (+ β-mercaptoethanol, 1:100).Homogenized lysate was transferred to gDNA eliminator spin.RNA was transferred to a RNeasy spin column and eluted into new tubes according to manufacturer's protocol.Total RNA concentration was measured using the NanoDrop ® ND-1000 (Thermo Scientific) and stored at − 80 °C until bulk RNA sequencing or quantitative real-time PCR.

Quantitative real-time PCR (qRT-PCR)
Coding DNA (cDNA) was generated using GoScript ™ Reverse Transcription System according to manufacturer's protocol.For the analysis of gene expression, the SSoFast ™ EvaGreen ® Supermix was mixed with respective primers (Additional file 1: Table S1) and amplified using the Light Cycler 480 system.For the analysis, gene expression levels were normalized to housekeeper genes Tyrosine 3-Monooxygenase (Ywhaz) and Glucuronidase Beta (Gusb).For qPCR, 4 animals per genotype were analyzed.

Bulk RNA sequencing
Samples were submitted to Genewiz GmbH (Leipzig, Germany) to prepare a standard and ultra-low input RNA-seq [40] library prior sequencing using an Illumina HiSeq platform.Reads were delivered as fastq files.Subsequent pre-processing and quality control of fastq files was performed by the Core Unit of Bioinformatics, Data Integration and-Analysis (CuBiDa) of the University Hospital Erlangen, Erlangen, Germany.

Bioinformatic analysis
In the first step of the bioinformatics data analysis of bulk RNA-seq data, the quality of the raw paired-end sequencing data sets (fastq) was assessed with the tool FastQC v0.11.9 [41].Samples that passed quality assessments were further optimized by a trimming step with the tool Trimmomatic v0.39 [42].Resulting samples were mapped with STAR aligner v2.7.9a [43] utilizing the mouse reference genome (Mus_musculus.GRCm39) from Ensembl release 104.The aligned sequences were converted to raw read counts per exon/gene with the tool featureCounts which is included in the Rsubread software package v2.14.2 [44].Gene symbol annotation was performed using the AnnotationDbi package [45].Modelling, normalization, and differential gene expression analysis was performed using the DESeq2 package [46] in combination with the R Version 4.1.2.Log2 fold-changes were set to > 1 and adj.p-values to 0.05.Differential expressed genes were pre-ranked by stats and gene set enrichment performed using the fgsea package.Results were visualized using the ggplot2 package.To determine cell lineages, the enricher-function of clusterProfiler package [47] was applied on the 50 most highly expressed transcripts.For assessment of cellular identity, the gene list was enriched on a single-cell RNA sequencing based mouse cell-marker dataset: CellMarker 2.0 [48].P value threshold was set to 0.05 and p value adjustment was performed using Benjamini-Hochberg (BH) procedure.Results were visualized using ggplot2 package.

Statistical analyses
Data were analyzed and visualized using GraphPad Prism 9.5.0 and CorelDraw X6 software.Shapiro-Wilk test was performed for determination of normal distribution of expression levels and cell counts.Welch's t-test was conducted for determination of statistical significance.If no Gaussian distribution was determined, statistical analysis was performed using Mann-Whitney-U test.Unless otherwise stated, all graphs represent mean values and standard deviation.P values were defined as statistically significant at p < 0.05.

Severe astrocytic response in the cortex, the putamen, and the substantia nigra of MSA-P patients
The upregulation of GFAP is indicative for the response of astrocytes to physiological or pathological stimuli [1].Based on a previous study by Hoffmann and colleagues [31] observing severe microgliosis in putaminal white matter of MSA patients and MBP29hα-syn mice, we assessed GFAP reactivity exclusively in grey matter of the putamen.GFAP + branched reactive astrocytes were increased in the motor cortex, the putamen, and the substantia nigra of MSA-P patients compared to age-and sex-matched controls (Fig. 1A,  B).The proportional highest increase of GFAP + reactive astrocytes was observed in the substantia nigra (2.8-fold; p < 0.0001) followed by the putamen (grey matter, 2.5-fold; p = 0.0003) and the motor cortex (precentral gyrus, 2.4-fold; p = 0.002).
Astrocytes are neural cells predominantly expressing high-affinity glutamate transporters involved in regulating glutamate levels within the synaptic cleft.Therefore, we asked whether astrocytic expression levels of the excitatory amino acid transporter-2 (EAAT2; mouse homologue GLT-1) are altered in the cortex and the putamen of MSA-P patients (Fig. 1B, upper panel).Interestingly, we observed a decreased number of GFAP + /EAAT2 + astrocytes in the precentral gyrus of MSA patients (p = 0.0571).However, we did not observe changes in GFAP + /EAAT2 + in the putamen of controls and MSA-patients (p = 0.6286, lower panel).Although no differences in the expression levels were observed, EAAT2 + astrocytes in MSA-P patients display accumulations of EAAT2 towards the nucleus indicating changes in distribution of EAAT2 in MSA.Due to the small sample size the quantification of the nuclear and diffuse distribution pattern did not reach statistical significance, but hints toward the nuclear distribution in the putamen of MSA-P patients (Additional file 6: Fig. S5).These findings of changed expression pattern of EAAT2 in the cortex and the putamen of MSA-P patients suggests impairments in essential functions of astrocytes caused by MSA-related pathology.

Pronounced astrocytic response in the cortex, the striatum, and the substantia nigra of MBP29-hα-syn mice
Based on the post mortem findings in MSA-P patients, we characterized the astrocytic response in MBP29-hαsyn mice at 4 weeks of age, an early (pre-motor) stage of this model [30].First, we confirmed the presence of alpha-synuclein (aSyn) pathology and myelination deficits in MBP29-hα-syn mice at 4 weeks of age (Additional file 5: Fig. S4).Furthermore, we quantified the number of GFAP + astrocytes in the cortex (pure grey matter) and the clinically most affected regions of the MBP29hα-syn mice, the striatum (mixed grey and white matter) as well as the substantia nigra (pars compacta, grey matter; Fig. 2B).A significant astrocytic response was present in the cortex, the striatum, and the substantia nigra of MBP29-hα-syn mice compared to NTGs (Fig. 2C, left panel).The proportional highest increase of GFAP + astrocytes was observed in the striatum (fourfold; p < 0.001) followed by the substantia nigra (3.5fold; p < 0.001) and the motor cortex (2.5-fold; p < 0.001; Fig. 2C, right panel).
ALDH1L1 represents a pan-astrocyte marker labeling astrocyte independently of the expression of GFAP [15].To exclude that the observed increase in GFAP expression is solely based on the overall increase of astrocytes, we calculated the ratio of GFAP + and ALDH1L1 + for both genotypes respectively (Additional file 3: Fig. S2).By analyzing the number of astrocytes expressing ALDH1L1 in these regions, we observed an overall elevated number of ALDH1L1 + astrocytes only in the cortex of MBP29hα-syn mice compared to NTGs (> 1.5-fold) (Additional file 3: Fig. S2).These results confirm that the increased number of GFAP + cells is not predominantly due to an increased recruitment of astrocytes or new-born astrocytes, but rather GFAP expression is triggered by the region-specific microenvironment.
To confirm the increased GFAP protein levels in astrocytes in MBP29-hα-syn, we performed qPCR analyses of cortical and striatal tissue homogenates (Fig. 2D).GFAP mRNA was increased in the cortex and striatum by 2-fold (p = 0.001) and 2.5-fold (p = 0.006), respectively.In contrast, mRNA levels of VIM, an important intermediate filament of astrocytes, was significantly increased in the striatum only (2.5-fold, p = 0.002).Furthermore, we focused on the gene expression levels of the glutamate reuptake transporters GLT-1 and GLAST to assess altered expression Fig. 1 Astrogliosis in post mortem brain tissue of MSA patients.A Representative image of DAB staining of GFAP + astrocytes in precentral gyrus, putamen, and substantia nigra of MSA-P patient (female, 67) and control individual (female, 60) and quantification of GFAP + cells/mm 2 (4 MSA patients vs. 4 Controls).SN was identified by presence of neuromelanin-containing neurons (white asterisks).Welch's t-test was used for statistical analysis.Scale bar = 20 µm.All three regions display elevated numbers of GFAP + astrocytes in cortex (p = 0.002), putamen (p = 0.0003), and substantia nigra (p < 0.0001) of MSA patients.CTRL = Control, MSA = multiple system atrophy, SN = substantia nigra.(B, Upper panel) Immunofluorescence staining of four MSA-P patients and three controls.For visualization of astrocytes GFAP was used as a marker (orange).To analyze expression of glutamate reuptake transporter tissue was stained for EAAT2 (green).Scale bar = 50 µm.(B, Lower panel) Overview of single cells expressing GFAP, EAAT2, and GFAP/EAAT2 (yellow, lower panel).Astrocytic GFAP/EAAT2 expression is decreased in the precentral gyrus (p = 0.0571).Moreover, a re-distribution towards the cytoplasm of EAAT2 is observed in astrocytes of MSA patients (Lower right panel) (See figure on next page.) of transcripts essential for astrocytic functionality.We did not detect differences in RNA levels for GLAST (cortex: 0.8-fold, p = 0.36; striatum: 0.75-fold, p = 0.2) and GLT-1 (cortex: 0.85-fold, p = 0.2; striatum: 0.83fold, p = 0.13).

Striatal astrocytes display a proinflammatory phenotype accompanied by transcriptional downregulation of important homeostatic functions in MBP29-hα-syn mice
To further evaluate the presence of astrocyte markers that relate to different astrocyte subsets in the cortex and the striatum, we determined GFAP and VIM levels in both regions by western blotting (Fig. 3A, upper left panel).Indeed, striatal GFAP levels were significantly elevated by 8.2-fold in MBP29-hα-syn mice (p < 0.001) and to a lower degree in the cortex (4.4-fold; p < 0.001).The protein levels of VIM were similarly increased in both regions of the MBP29-hα-syn mice, but at smaller magnitude compared to GFAP (cortex: twofold, p = 0.0065; striatum: 2.5-fold, p < 0.001).
Highly pure ATP1B2 + (ACSA2 + ) population by additional magnetic bead removal of O4 + -cells Our observation of differential expression of several astrocyte-associated transcripts and proteins in the striatum and cortex of MBP29-hα-syn mice prompted us to perform RNA sequencing of astrocytic populations.To this end, we designed a recent astrocyte isolation protocol using MACS via ATP1B2 sorting.Following cell isolation using ACSA2-MicroBeads, we still detected a higher number of O4 + -cells (> 30%).We removed O4 + -cells by an additional pre-sorting step labeling for O4 + -cells.After FcR blocking, we incubated the cell suspension using anti-O4-Microbeads.Subsequently, we performed an anti-ACSA2-MicroBeads labeling step followed by the flow-through of the O4 population.To assess purity and viability after the two-step MACS approach, we performed flow cytometry (FC) analysis demonstrating a high yield for ACSA2 + cells (86.3%) in conjunction with a significant reduction of O4 + cells down to 5% without inducing cell death (Fig. 3B, left panel).For assessment of cell identity, complementary to the results of the flow cytometry analysis, we further applied the enricherfunction of the clusterProfiler-package to merge the top 40 most highly expressed transcripts of the cortical and striatal datasets on a cell type annotation database [48].
We confirmed the identity of the isolated cell population as astrocytes (Fig. 3B, right panel).In addition, isolated cells express markers of neural stem cells and Bergmann glia known to share astrocytic transcriptional patterns (Table 3).

Transcriptional upregulation of pro-inflammatory transcripts accompanied by altered homeostasis-associated transcripts in the striatum of MBP29-hα-syn mice
Hypothesizing that the striatum presents a pro-inflammatory, hostile microenvironment, our next objective was to elucidate the specific transcriptional profile of isolated ACSA2 + astrocytes.Given the limited volume of the murine striatum and consequently the low RNA concentration, we employed a novel ultra-low amount sequencing technique.To ensure comprehensive transcriptomic data sets, we initially performed principal component analysis (PCA) to assess samples based on genotype clustering (Additional file 4: Fig. S3).A clear separation and clustering were present between both genotypes indicating a prominent divergence of transcriptional profiles and molecular signatures.
To obtain a more detailed overview of transcript enrichment in specific pathways, we applied functional  (See figure on next page.)Fig. 4 Transcriptome analysis of striatal astrocytes after MACS via ACSA2 of MBP29-hα-syn mice.A Mosaic plot of altered astrocytic functions in the striatum of MBP29-hα-syn mice based on existing dataset assessed by Zamanian et al. [16].Astrocytes display a distinct upregulation of transcripts linked to astrocyte reactivity.Notably, there is a strong enrichment in cytokine activity, extracellular matrix (ECM) organization, metabolic processes and transcription factor activity, as well as not clearly classifiable functions (Miscellaneous).B Volcano plot of differentially expressed genes (DEGs).DEGs were analyzed comparing MBP29-hα-syn mice with non-transgenic littermates.Thresholds were set to log2foldchange > 2.0 and adj.p value < 0.05.Y-axis: negative decade logarithm of adj.p value.X-axis: log2foldchange of gene expression levels.In turquoise: significantly downregulated genes, in grey: genes without statistical significance, in red: significantly upregulated genes.Top hits were marked with official gene symbols by the HUGO Gene Nomenclature Committee (HGNC).C, D Heatmap of the 20 most highly up-and downregulated genes according to adjusted p-value.Gene expression was scaled by rows to visualize differences in gene expression between and within the genotypes using log2foldchange of gene expression.Striatal astrocytes demonstrate a profound upregulation of pro-inflammatory transcripts.Additionally, homeostatic functions, such as lipid metabolism, calcium transport, and neurotransmitter signaling are impaired in astrocytes on transcriptional level.E, F Functional gene set enrichment analysis of DEGs (FDR > 0.05).Pathways are ordered by normalized enrichment score (NES).Nomenclature of pathways is modified from official hallmark and biological processes pathways.Bold: Pathways associated with pro-inflammatory pathways and impaired homeostasis gene set enrichment analysis (fgsea) on two different pathway sets: hallmark gene set for a generalized overview and KEGG for a more specific annotation.Our results were consistent with the initial analysis based on expression levels of the 20 differentially up-/downregulated genes, respectively (Fig. 4E, F).We detected a substantial enrichment of transcripts, particularly in interferon-α (NES = 2.6), complement (NES = 2.3), and interleukin-2/STAT5 signaling (NES = 1.97) confirming the pro-inflammatory signature of striatal astrocytes.
In conjunction with the pro-inflammatory qPCR findings in cells of striatal homogenates, it suggests that ACSA2 + astrocytes may drive neuroinflammatory processes within the striatum of MBP29-hα-syn mice.Moreover, striatal ACSA2 + astrocytes of transgenic mice show signs of an impaired lipid homeostasis and energy supply further implying astrocytic malfunctioning.

Cortical astrocytes adopt an anti-inflammatory, secretory phenotype in MBP29-hα-syn mice
To characterize the transcriptional signature of cortical astrocytes, we performed an identical analysis as described for the striatum.The general count of differentially expressed transcripts based on previously published reactivity markers is much lower in cortical ACSA2 + astrocytes (Table 5).Furthermore, there is no association of transcripts linked to cytokine-or inflammatory responses in the isolated astrocytic population [16].Interestingly, the sole transcript linked to immune response is lipocalin 2 (LCN2) showing a downregulation in cortical ACSA2 + astrocytes of MBP29-hα-syn mice.
For further characterization of cortical ACSA2 + astrocytes, we applied fgsea.Cortical astrocytes exhibit a rather secretory phenotype by upregulation of transcripts associated with protein secretion (Fig. 5E).In contrast to striatal ACSA2 + astrocytes, the overall inflammatory response was reduced in cortical astrocytes of MBP29hα-syn mice, consistent with findings of the 20 most highly downregulated genes (Fig. 5E, F).Detailed analysis of biological processes revealed upregulated transcripts involved in receptor-mediated endocytosis and amyloid beta metabolic processes (Fig. 5F).The enrichment of downregulated pathways observed further supports the notion of particular immune system-associated processes being downregulated, as well as processes involved in sequestration of triglycerides suggested to be protective in lipotoxic conditions.In conjunction with findings obtained from striatal ACSA2 + astrocytes, it is evident that cortical and striatal ACSA2 + astrocytes exhibit a very distinct molecular pattern despite a similar degree of GFAP upregulation.

Region-specific upregulation of pro-inflammatory transcripts in ACSA2 + astrocytes of MBP29-hα-syn mice
To better delineate differences between striatal and cortical ACSA2 + astrocytes of MBP29-hα-syn mice, we conducted an analysis of differentially expressed genes (log2 fold change > 1.0, padj < 0.05) followed by gene set enrichment analysis (FDR < 0.05; Fig. 6A).In respect to the separate analyses of both brain regions, this approach confirmed that pro-inflammatory pathways (TNF signaling via Nfkb, interferon γ response, and inflammatory responses) are predominantly present in striatal ACSA2 + astrocytes.Merging both regional datasets demonstrates the clearly distinguishable transcriptomic profile of cortical and striatal astrocytes with a low total number of overlapping DEGs of 79 genes shown by Venn-diagram (Fig. 6B).Performing overrepresentation analysis (ORA) to display the non-overlapping genes (942 striatal vs 573 cortical transcripts) of both datasets, we confirmed that striatal astrocytes mainly exhibit an inflammationassociated molecular profile, regulate migration and cell adhesion, and responds to external stimuli (Fig. 6B, lower panel).Inversely, cortical astrocytes appear to be involved in supportive processes, such as axonogenesis, myelination, gliogenesis, and differentiation processes (Fig. 6B, upper panel).This ORA highlights the diversity Fig. 5 Transcriptome analysis of cortical astrocytes after MACS via ACSA2 of MBP29-hα-syn mice.A Mosaic plot of altered astrocytic functions in the cortex of MBP29-hα-syn mice based on existing dataset [16].Reactivity profile of cortical astrocytes show less pronounced upregulation of reactivity-associated genes compared to striatal astrocytes.Primary processes affected refer to metabolic and ECM organization, as well as unclassified processes.B Volcano plot of DEGs analyzed comparing MBP29-hα-syn mice with non-transgenic litter mates.Thresholds were set to log2foldchange > 2.0 and adj.p value < 0.05.Y-axis: negative decade logarithm of adj.p value.X-axis: log2foldchange of gene expression levels.In turquoise: significantly downregulated genes, in grey: genes without statistical significance, in red: significantly upregulated genes.Transcripts with most significant adj.p-values and log2foldchange were visualized using official gene symbols by HGNC.C, D Heatmap of the 20 most highly up-and downregulated genes according to adjusted p value.Gene expression was scaled by rows to visualize differences in gene expression between and within the genotypes using log2foldchange of gene expression.Cortical astrocytes display significant upregulation of transcripts associated with oligodendrocyte development and myelination (ERMN, MAG, MYRF, OPALIN), ion and neurotransmitter homeostasis, and neuroprotection (SLC6A7, TLL2, SLCA24A2, SIS).E, F Functional gene set enrichment analysis of DEGs (FDR > 0.05).Pathways are ordered by normalized enrichment score (NES).Nomenclature of pathways is modified from official hallmark and biological processes pathways.E Transcripts enriched in protein secretion are upregulated in cortical astrocytes, whereas a significant downregulation of transcripts enriched in pro-inflammatory processes (IL-6/STAT3, complement, IFNγ, IFNα) was observed.F Upregulated transcripts demonstrate enrichment in receptor-mediated endocytosis and amyloid-β metabolism.Enrichment of downregulated transcripts mostly in immune system associated processes (complement, dendritic cell activation) confirms pattern already observed in hallmark analysis (See figure on next page.) of cortical and striatal astrocytes regarding their profile and potentially associated functions in the context of MSA-related pathology.Overall, we show that striatal astrocytes exhibit an increased immune response compared to cortical astrocytes.

Oligodendroglia-associated transcripts are present within close proximity of astrocytic processes
As we observed a profound upregulation of pro-myelinogenic and oligodendroglial transcripts in cortical ACSA2 + astrocytes, we asked whether we are able to detect oligodendrocyte-associated transcripts in astrocytes in MBP29-hα-syn mice.To this end, we applied in-situ hybridization by RNAscope ISH Technology to detect oligodendroglial SRY-Box Transcription Factor 10 (SOX10) and MYRF transcripts in cortical GS + astrocytes (Fig. 7).Indeed, we clearly detected Sox10 and Myrf transcripts within GS + astrocytic processes.Due to the lack of markers to visualize the entire network of astrocytic processes, we were not able to trace the transcripts to the respective nuclei of astrocytes.Nevertheless, the spatial proximity of astrocytic processes and oligodendroglial transcripts suggests that oligodendroglial transcripts may be located in the processes of astrocytes in the cortex of MBP29-hα-syn mice.

Discussion
Reactive astrogliosis is a prominent feature in multiple neurodegenerative disorders [51].Several studies taking advantage of microarray-and RNA sequencing technology and surface marker-based sorting of astrocytes emphasized region-and context-dependent diversity of astrocytes [16,[33][34][35]52].Here, we analyzed GFAP expression as a proxy for reactive astrogliosis in post mortem tissue of MSA-P patients in brain regions severely affected by disease pathologies such as the motor cortex, putamen, and substantia nigra.We detected an increase of GFAP + cells suggesting reactive astrogliosis in all three regions.Notably, the putamen and the substantia nigra, most prominently affected in MSA-P, displayed a proportionally higher number of GFAP + astrocytes compared to motor cortex.To extend the characterization of astrocytes, we analyzed expression of EAAT2, an important transporter for glutamate reuptake.A decrease of glutamate reuptake transporter expression has been linked to other neurodegenerative disorders, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) [53][54][55][56].Quantifying GFAP + /EAAT2 + astrocytes hinted towards decreased numbers in MSA-P patients compared to controls and revealed altered distribution of EAAT2 in both brain regions.
Analyzing the expression of EAAT2 in the cortex and the putamen of MSA-P patients provided evidence of a lower number of GFAP + /EAAT2 + astrocytes in MSA-P patients.Notably, the distribution pattern of EAAT2 is altered in MSA-P patients in cortex and striatum, demonstrating a re-distribution from a diffuse to a nuclear pattern.This altered localization of EAAT2 was already described in vitro in a model of AD upon exposure of astrocytes to Aβ 1-42 [57].The altered expression pattern results in a slower clearance time of excessive glutamate in the synaptic cleft and is therefore potentially linked to Aβ-related pathology.Based on this astrocytic phenotype in the human post mortem tissue, we further characterized astrocytes in MBP29-hα-syn mice.Mirroring the findings in human post mortem tissue, we also observed a substantial increase of GFAP + cells in the cortex, the putamen and the substantia nigra.An enhanced astrocytic response was previously observed in grey compared to white matter regions of MSA-P patients and/or in MBP29-hα-syn mice [31].The similarities in GFAP expression between MSA patients and the present transgenic MSA mouse model prompted us to a comprehensive analysis of astrocytes in distinct brain regions.Interestingly, a tendency of an increased total astrocyte number was present in the cortex of MBP29hα-mice.Previous work showed an increased number of oligodendrocytes in the corpus callosum and preserved numbers of oligodendrocytes in the putamen of MBP29hα-mice [32].These findings are further supported by the observations of Ge and colleagues, hinting towards the presence of a glial progenitor population at the age between 3-4 weeks postnatally [58].The increased levels of the reactivity-associated markers GFAP and VIM in the striatum suggest that these astrocytes may be much more responsive to α-syn pathology compared to other regions.This notion was accentuated by a decrease of GLT-1 and GLAST protein expression exclusively in the (See figure on next page.)Fig. 6 Comparison of cortex-and striatum-derived astrocytes from MBP29-hα-syn mice.A Functional gene set enrichment analysis based on DEGs.Striatal transcripts were compared to cortical transcripts and subsequently enriched in hallmark pathways and biological processes.Most significant enrichment of striatal transcripts were observed in pathways associated with a pro-inflammatory response and energy homeostasis, implying a more pronounced reactivity in striatum compared to the cortex.B Venn-diagram of overlapping genes of striatal and cortical transcripts derived from displaying 79 shared differentially expressed genes.Overrepresentation analysis of the non-overlapping genes (973 striatal, 573 cortical genes) displaying the top 8 overrepresented pathways sorted by the count of enriched transcript.Color code is based on the adj.p-value striatum potentially resulting in glutamate excess.The context-dependent expression levels of glutamate transporter were described in 6-hydroxydopamine-lesioned PD model [59].Alternatively, the reduced levels of GLT-1 and GLAST in the striatum compared to the cortex hint towards the intrinsic heterogeneity between cortical and striatal astrocytes thus supporting the findings of a distinct profile of astrocytes in cortex and striatum of MBP29-hα-syn mice.Assessing the expression of additional homeostasis-associated astrocyte markers, we observed an upregulation of GS in the cortex while there was no significant change in the striatum.Lack of GS expression was already shown to impact neurodegenerative processes [60].Furthermore, AQP-4 and GAP-43 protein levels were more increased in the cortex compared to the striatum suggesting that the capacity to maintain CNS homeostasis of striatal astrocytes is potentially impaired in MBP29-hα-syn mice.
To investigate the impact of MSA-related neuropathological processes on astrocytes on a cellular level, we developed an astrocyte isolation method with high purity modified from an existing surface marker-based sorting protocol using ATP1B2 as an unbiased astrocyte-specific surface marker [61] and subsequent astrocyte RNA sequencing.The bioinformatic analysis of the gene expression patterns revealed astrocyte identity in the isolated cell population but also indicated the pattern of neural stem cells.On the one hand, this enrichment might result from the shared expression patterns of astrocytes and neural stem cells [62].On the other hand, since these mice are in a postnatal developmental period, this enrichment may be due to the presence of co-isolated neural stem cells residing in the subventricular zone close to the striatum [63].Improving the isolation approach, we were able to achieve a high purity of ACSA2 + cells and high-quality outcome after bulk RNA sequencing.To evaluate astrocyte reactivity, we compared the astrocytic gene expression profile derived from the cortex and striatum MBP29-hα-syn mice to RNA-seq datasets previously published by Zamanian et al. [16] which induced reactive astrogliosis in wild-type mice using intraperitoneal lipopolysaccharide injections.Interestingly, the transcriptome of both astrocyte gene expression profiles overlapped with Zamanian et al., however, striatal astrocytes displayed an enrichment of these markers.
The most striking result to emerge by analyzing the cortex and the striatum, was a pro-inflammatory signature in striatal compared to cortical astrocytes.Elevated levels of pro-inflammatory cytokines were observed in the cerebrospinal fluid (CSF) of MSA patients [64].Moreover, Valera and colleagues assessed pro-inflammatory cytokine levels in transgenic MSA mice [65].In this study, the authors provide evidence of elevated CSF levels of IL-1β, IL-1ra, IL-13, IL-17, MIP-1α, and G-CSF using a cytokine proteomic assay matching the findings of the present transcriptomic findings.Additionally, regiondependent responses have been described in mouse models of Huntington's disease [20,66].Striatal mouse astrocytes have been shown to respond context-specifically by G i -GPCR signaling in vivo with an upregulation Fig. 7 In-situ hybridization of oligodendrocyte-associated transcripts in the cortex of MBP29-hα-syn mice.RNAscope hybridization of oligodendroglial transcripts in astrocytes.GS was used as a pan-astrocyte marker (orange).Sox10 (cyan) and Myrf (yellow) were considered as oligodendrocyte markers covering characteristic of maturing and myelinating oligodendrocytes.Nuclei were counterstained using DAPI (blue).In MBP29-hα-syn mice, oligodendroglial co-label with astrocytic processes stained with GS, but are not unequivocally localized within the processes.Scale bar = 20 µm of neuroinflammatory responses but whether the inflammatory response is toxic or protective remained unclear [66].There is evidence that striatal astrocytes demonstrate altered transcriptomic profiles in inflammatory environment [67] associated with impaired homeostatic functions for calcium and glutamate signaling [68].Distinct populations of astrocytes were identified in a HD mouse model exhibiting variable transcriptional phenotypes in the cortex reflecting a rather protective phenotype consistent with our findings [69,70].Another study characterizing astrocytes by single nuclei sorting in the prefrontal cortex, most severely affected in AD patients, revealed decreased homeostatic and increased levels of inflammatory transcripts [71].In general, comparing astrocytes derived from AD patient's post mortem brains, revealed non-homogenous astrocyte population including subpopulations capable of upregulating supportive processes as stress and survival response, but also subsets with metabolic stress and defective clearance [72].Another remarkable result is that differentially expressed genes in striatal astrocytes are enriched in downregulated lipid-associated pathways and impaired energy homeostasis in the cortex of hereditary spastic paraplegia and AD patients [73,74].Taken together, a more inflammatory astrocytic response is present in CNS regions predominantly affected by the respective pathology.
Here, we assessed a pronounced pro-inflammatory state of astrocytes accompanied by impaired homeostatic processes via regional isolation and subsequent bulk RNA sequencing.The alteration of electrophysiological characteristics in neuroinflammatory conditions induced by brain abscesses has already been described in striatal astrocytes [75,76].Furthermore, an induction of neurotoxic astrocytes via release of microglial TNFα and IL1β has recently been shown [11].In contrast, astrocytes are the major source of chemokines (CCL2, CXCL1 [77]) with corresponding receptors expressed by microglia potentially implicating a bi-directional communication.The upregulation of macrophage colonystimulating factor (CSF-1) in astrocytes upon exposure to pro-inflammatory cytokines was observed which in turn suppressed microglial interferon-and MHC expression [78].Another potential route of astrocyte-microglia communication is the microglial secretion of vascular endothelial growth factor β (VEGFB) for modulation of pathogenic characteristics of astrocytes [79].In addition to the secretory component of astrocytes, components of the innate immune system play a pivotal role, such as tolllike receptors (TLR) and complement factors [80][81][82], which were observed to be upregulated in the striatum of MBP29-hα-syn mice.Especially, TLR4 is known as an essential receptor for astrocyte and microglia activation by aSyn [83] and could therefore play a crucial role in the crosstalk of both cell types.More detailed analysis of the complex interaction between microglia and astrocytes is necessary in future work to reveal potential mechanisms of cross-talk and maintenance of immune response in the CNS.
To our surprise, cortical astrocytes display an oligodendroglial and pro-myleinogenic expression pattern within the 20 most highly upregulated genes (ERMN, MAG, OPALIN, MYRF).Given the fact, that glutamate transporter expression is influenced by MSA pathology, we are not able to apply more specific isolation via the glutamate reuptake transporter GLAST (ACSA1).Nonetheless, we were able to determine high enrichment of astrocytes by bioinformatical enrichment algorithm.Moreover, we applied an RNA hybridization approach to confirm the presence of oligodendroglial transcription factors within or in proximity of astrocytic processes.A second limitation is due to the fact that the visualization of an entire astrocyte is difficult using commercially available antibodies against astrocyte markers, such as GFAP, ALDH1L1, S100β, and GS.Using RNAscope, we succeeded to visualize the presence of oligodendroglial transcripts in proximity of astrocytic processes counterstained with GFAP or GS.Combined with the enrichment of upregulated transcripts in cortical astrocytes involved in protein secretion, these results may potentially suggest that astrocytes support oligodendrocytes by energy supply, secreting trophic factors, and support of immune response as shown in other demyelinating diseases [84][85][86][87].Furthermore, intercellular RNA transfer might serve as a possible mechanism for astrocytes to provide support to neighboring cells [88].Future investigations are necessary to fully explore the specific potential of astrocytes to endogenously express distinct oligodendroglial transcripts thereby either promoting a change of cellular identity or supporting dysfunctional oligodendrocytes in the cortex of MBP29-hα-syn mice.
The present study provides a detailed region-specific characterization of the transcriptomic landscape of astrocytes in MSA.Our work contributes to a better understanding of astrocyte heterogeneity in distinct brain regions differentially affected by MSA-related neuropathology.The upregulation of reactivity-associated proteins in astrocytes, to certain limits, does not necessarily affect astrocytic capacity to support surrounding cells or the maintenance of CNS homeostasis in the affected regions.Moreover, despite reactive cortical astrogliosis, MBP29-hα-syn mice upregulate homeostasis related proteins such as AQP-4, GS, and GAP-43.In the striatum, severely affected by MSA pathology, we provided evidence for the presence of reactive astrocytes acquiring a more harmful phenotype by upregulation of pro-inflammatory transcripts • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

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Table 1
Overview of MSA-P patients with clinical characteristics and controls

Table 2
Overview of astrocytic proteins involved in CNS homeostasis and their associated functions

Table 3
Assessment of cell identity using clusterProfiler

Table 4
GO-Terms and associated transcripts differentially expressed in striatal astrocytes of MBP29-hα-syn mice

Table 5
GO-Terms and associated transcripts differentially expressed in cortical astrocytes of MBP29-hα-syn mice